1. Field of the Invention
The present application relates to making a chimeric molecule that is used to prevent blood vessel formation as a form of treating cancer or treating eye disease.
2. Description of the Background
Vascular endothelial growth factor-A (VEGF-A) is a critical regulator of tumor angiogenesis, mainly through the activation of its primary receptor, VEGF receptor 2 (VEGFR2). VEGF-A is expressed in most tumor cells and corresponding stromal cells throughout every stage of tumor progression, while VEGFR2 is highly expressed in growing tumor vessels, leading to the formation of structurally and functionally malformed tumor blood vessels. VEGF-A specifically binds to the second immunoglobulin (Ig) homology domain (D2) of the extracellular region of VEGFR2, resulting in activation of pro-angiogenic signalling. For the past decade, much effort has been devoted to targeting this VEGF-A/VEGFR2 signalling pathway using monoclonal antibodies, soluble decoy receptor fusion proteins, and small molecular inhibitors in cancer patients. While the current therapeutic blockade of VEGF-A/VEGFR2 signalling provides clinical benefits, the anti-cancer effect is modest and transient, eventually giving rise to acquired resistance through the activation of alternative pro-angiogenic pathways and further recruitment of pro-angiogenic cells such as tumor-associated macrophages (TAM). These limitations highlight current unmet needs in anti-angiogenic cancer treatment strategies, which must be addressed for successful therapy development.
Many ocular diseases are also associated with abnormal angiogenesis and up-regulated VEGF. Particularly, exudative age-related macular degeneration (AMD) is one of the most important causes of blindness in developed countries and the most clinically critical subtype of AMD. In exudative AMD, the macular region rapidly deteriorates due to abnormal angiogenesis, termed “choroidal neovascularization (CNV)” which arises from the choriocapillary across the retinal pigment epithelium (RPE) and Bruch's membrane to subretinal space of macula. CNV is also a major complication that threatens the vision of patients with various retinal degenerative and inflammatory diseases, including pathologic myopia and ocular histoplasmosis. Angiogenesis is normally a compensatory mechanism of our body in pathologic situations such as coronary collateral formation and wound healing process. And this mechanism is also triggered by an oxygen insufficiency state as known as “hypoxia”. Hypoxic state stimulates hypoxic inducible factors (HIFs) including VEGF and it plays a crucial role in angiogenesis. In addition, eyes with high concentration of VEGF suffer from leakage from retinal vessels, and subsequently, macular edema develops. Therefore, VEGF may be a therapeutic target for ocular diseases associated with abnormal angiogenesis and vascular leakage such as exudative AMD, diabetic retinopathy, retinopathy of prematurity, neovascular glaucoma, corneal neovascularization, retinal vein occlusion and macular edema due to diabetic retinopathy or retinal vein occlusion.
VEGF-A binds to both VEGFR1 and VEGFR2. The binding affinity of VEGFR1 to VEGF-A (<10˜20 pM) is much higher than that of VEGFR2 (<100˜125 pM). In addition, VEGFR1 is a receptor for other pro-angiogenic ligands, VEGF-B and placental growth factor (PlGF), which have recently been highlighted as alternative targets for anti-angiogenic therapy. Because of its ability to bind multiple pro-angiogenic ligands, VEGFR1 has been considered as a potential backbone for the development of a novel decoy receptor fusion protein for therapeutic purposes. However, the efficiency of a decoy receptor fusion protein which consisted of the first 3 Ig domains of VEGFR1 fused with the Fc region of IgG1 (VEGFR1-Fc) proved unsatisfactory, due to non-specific binding to the extracellular matrix (ECM) attributed to the abundant positively charged residues in the third Ig domain (VEGFR1 D3) and its high isoelectric point (pI) value. Nonetheless, this finding inspired the invention of VEGF-Trap (Aflibercept from Regeneron), consisting of VEGFR1 D2 and VEGFR2 D3 fused to IgG1 Fc. By switching VEGFR1 D3 to VEGFR2 D3, the net pI of VEGF-Trap was decreased, resulting in less ECM binding and an improved pharmacokinetic (PK) profile compared to VEGFR1-Fc. However, because VEGFR2 D3 was used instead of VEGFR1 D3, the high-affinity binding of VEGF-A and PlGF was disturbed. Hence, the important issue to be addressed now is how to incorporate VEGFR1 D3 into a decoy receptor while minimizing non-specific ECM binding.
Glycosylation is a post-translational modification that results in the addition of carbohydrate chains to specific asparagine (N-linked glycosylation) or serine/threonine (O-linked glycosylation) residues. Glycosylation of secreted and membrane proteins affects their biochemical and biological properties. It usually provides a negative charge and increases solubility, thus diminishing non-specific binding to the ECM. Moreover, glycosylation grants resistance to proteolysis and extended serum half-life, enhancing a protein's PK profile. Glyco-engineered therapeutic proteins such as Aranesp (erythropoietin) from Amgen and Gazyva (obinutuzumab) from Genentech are good examples that exploited these advantages.
Here, we developed a novel VEGF decoy receptor fusion protein, VEGF-Grab. Parental VEGFR1-Fc (VEGFR1 D2-D3 fused to Fc) was used as a backbone, and new potential glycosylation sites were introduced into the positively-charged patch of VEGFR1 D3 by site-directed mutagenesis. This engineered VEGF-Grab showed significantly improved decoy efficiency and a dramatic decrease in net pI, thus attenuating non-specific ECM binding and enhancing PK profiles. Thus, VEGF-Grab strongly suppressed tumor angiogenesis, progression, and metastasis via effective capturing of three VEGFR1 ligands, VEGF-A, VEGF-B, and PlGF. Furthermore, we show the intravitreal therapeutic efficacy of VEGF-Grab3 to regress new vessels in laser-induced CNV and oxygen-induced retinopathy (OIR) murine models, which are reliable methods in predicting the therapeutic value of anti-VEGF therapies now approved for treating AMD and diabetic retinopathy.